MRNA composition and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and relates to an mRNA composition and application thereof, the mRNA composition comprises lipid nanoparticles and mRNA contained in the lipid nanoparticles, and the mRNA can express CXCL3. After being infused through caudal vein, the mRNA composition disclosed by the invention can chemotaxis a large number of mononuclear macrophages to infiltrate a mouse sclerotic liver and phagocytize fibrosis, so that the fibrosis level in the mouse liver is remarkably reduced. Experimental results show that the composition almost has no toxicity to normal tissues, is relatively high in safety, and shows the potential in treatment of hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an mRNA-lipid nanoparticle complex and a preparation method and application thereof. Background Art

[0002] In the context of chronic liver damage such as long-term viral infection, alcoholism, high-fat diet, and autoimmune diseases, excessive deposition of the extracellular matrix (ECM) leads to liver fibrosis. Liver fibrosis is an inevitable path for various chronic liver diseases, and continued progression will develop into cirrhosis and even liver cancer. Inhibiting or even reversing liver fibrosis and cirrhosis has always been a hot topic in the field of liver disease research, but no significant progress has been made so far. Current treatment strategies for liver fibrosis can only delay the process of fibrosis, but cannot promote ECM degradation to reverse fibrosis. Therefore, there is an urgent need to explore new treatments for liver fibrosis.

[0003] There are a large number of macrophages in the liver. Currently, a large number of basic experimental studies have shown that macrophages play a vital role in the reversal of endogenous liver fibrosis. However, cell therapy programs that directly infuse macrophages usually have disadvantages such as long cycles, strict process requirements, and high treatment costs, making them difficult to be applied and promoted.

[0004] mRNA therapy has become a research hotspot in the field of drug development. mRNA produces corresponding proteins through translation and exerts biological functions. In theory, mRNA can replace existing protein-level drugs. mRNA therapy is particularly suitable for treating diseases that do not require continuous and lifelong expression of therapeutic proteins. It has the advantages of easy large-scale production and no potential risk of insertion mutations. Summary of the invention

[0005] One of the purposes of the present disclosure is to provide an mRNA-lipid nanoparticle complex for treating liver cirrhosis in a safe and effective manner by promoting macrophage infiltration and degrading fibrosis.

[0006] In order to achieve the above objectives, the present disclosure adopts the following technical solutions:

[0007] In a first aspect, the present disclosure provides an mRNA composition comprising mRNA and lipid nanoparticles, wherein the mRNA is contained in the lipid nanoparticles, and wherein the mRNA is capable of expressing chemokine ligand 3 (CXCL3).

[0008] In some embodiments, the amino acid sequence of CXCl3 is shown in SEQ ID NO: 2. In some embodiments, the mRNA comprises a CXCL3 coding region, and the CXCL3 coding region comprises a nucleotide sequence shown in SEQ ID NO: 5 or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO: 5.

[0009] In some embodiments, the nucleotide sequence of the CXCL3 coding region is shown in SEQ ID NO:5.

[0010] In some embodiments, the mRNA further comprises a 5' cap structure, a 5' UTR, a Kozak sequence, a 3' UTR and / or a poly A region.

[0011] In some embodiments, the 5'UTR sequence is the sequence shown in SEQ ID NO:6.

[0012] In some embodiments, the 3'UTR sequence is the sequence shown in SEQ ID NO:8.

[0013] In some embodiments, the mRNA comprises the nucleotide sequence shown in SEQ ID NO:4 or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO:4; in some preferred embodiments, the nucleotide sequence of the mRNA is as shown in SEQ ID NO:4.

[0014] In some embodiments, the lipid nanoparticles comprise an ionizable lipid, a non-cationic lipid, cholesterol, and a polyethylene glycol (PEG) lipid.

[0015] In some embodiments, the ionizable lipid is selected from (4-hydroxybutyl) aminobis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315, CAS No.: 2036272-55-4), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)aminobis(dodecan-2-ol) (C12-200), 4-(N,N-dimethylamino)butyric acid-6,9,28,31-tetraene-19-triacontanoate (MC3), 1,2-linoleyloxy-N,N-dimethyl-3-aminopropane (DLinD MA), 2,2-linoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2DMA), (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(1H-imidazol-4-yl)propanoic acid ethyl ester (ICE), (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadecadien-9,12-dien-1-yl)tetracosyl-15,18-dien-1-amine (HGT500 0), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecadien-1-yl)tetracosine-4,15,18-triethylenetetramine-1-amine (HGT5001), 3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1-yl]amino]butyl]-2,5-piperazinedione (OF-02), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl ammonium trifluoroacetate (DOSPA), 5-carboxysperminylglycine-bisoctadecylamide (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-(1,2-dimyristoyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybut-4-oxy)-1-(cis, cis-9,1-(cis-1-2-octadeceneoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentyloxy]-3-dimethyl-1-(cis,cis-9',1-2'-octadeceneoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1, 2-N,N'-dilinoleylcarbamyl-3-dimethylpropylamine (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2-((2,3-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethylamine (HGT4003) One or more. In some preferred embodiments, the ionizable lipid is ALC-0315. ,

[0016] In some embodiments, the non-cationic lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimide) In some preferred embodiments, the non-cationic lipid is distearoylphosphatidylcholine.

[0017] In some embodiments, the PEG lipid is selected from one or more of PEG-phospholipids and PEG-ceramides, for example, selected from methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159), dimyristoylglycerol-PEG2000 (DMG-PEG2000), distearoyl phosphatidylethanolamine-PEG2000 (DSPE-PEG2000), dipalmitoyl phosphatidylethanolamine-PEG2000 (DPPE-PEG2000), distearoyl phosphatidylethanolamine-PEG2000 (DMPE-PEG2000), dioleoyl phosphatidylethanolamine-PEG2000 (DOPE-PEG2000), distearoyl phosphatidylethanolamine-PEG1000 (DSPE-PEG1000). 00), dipalmitoylphosphatidylethanolamine-PEG1000 (DPPE-PEG1000), dimyristoylphosphatidylethanolamine-PEG1000 (DMPE-PEG1000), dioleoylphosphatidylethanolamine-PEG1000 (DOPE-PEG1000), distearoylphosphatidylethanolamine-PEG550 (DSPE-PEG550), dipalmitoylphosphatidylethanolamine-PEG550 (DPPE-PEG550), dimyristoylphosphatidylethanolamine-PEG550 (DMPE-PEG550), PEG-cholesterol, PEG2000-ceramide, PEG1000-ceramide, PEG750-ceramide, PEG550-ceramide, one or more thereof. In some embodiments, the PEG lipid is ALC-0159.

[0018] In some preferred embodiments, the lipid nanoparticles comprise ionizable lipid ALC-0315, distearoylphosphatidylcholine, cholesterol, and PEG lipid ALC-0159.

[0019] In some embodiments, the particle size of the lipid nanoparticles is 85-120 nm, for example, it can be 85 nm, 86 nm, 87 nm, 88 nm, 90 nm, 92 nm, 94 nm, 96 nm, 98 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, preferably 88-89 nm, and more preferably 88 nm.

[0020] In some embodiments, the monodispersity of the lipid nanoparticles is 0.030-0.99, for example, it can be 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.063, 0.065, 0.067, 0.070, 0.073, 0.075, 0.077, 0.080, 0.083, 0.085, 0.087, 0.090, 0.095, 0.099, preferably 0.065-0.085, and more preferably 0.075.

[0021] In some embodiments, the zeta potential of the lipid nanoparticles is 3.0-8.0 mV, for example, 3.0 mV, 3.5 mV, 4.0 mV, 4.5 mV, 5.0 mV, 5.3 mV, 5.5 mV, 5.7 mV, 5.9 mV, 6.0 mV, 6.1 mV, 6.3 mV, 6.5 mV, 7.0 mV, 7.5 mV, 8.0 mV, preferably 5.5-6.5 mV, and more preferably 6.1 mV.

[0022] In some embodiments, the composition is prepared as follows:

[0023] An expression cassette for expressing CXCL3 protein was inserted into the pUC57-Kan plasmid, which contained from 5' to 3' end: a promoter site for T7 RNA polymerase, 5' UTR, "Kozak sequence + DNA sequence of CXCL3 shown in SEQ ID NO: 1", 3' UTR, poly A and a cleavage site for BspQI restriction endonuclease.

[0024] After amplification, the BspQI restriction endonuclease was used for linearization, and the in vitro transcription template expressing CXCL3 was recovered. ARCA (NEB, S1411S) was added to cap the in vitro transcription to obtain CXCL3 mRNA.

[0025] Encapsulation of CXCL3 mRNA: The liposomes contain ionizable lipids, non-cationic lipids, cholesterol and PEG-modified lipids. The ionizable lipid ALC-0315, the non-cationic lipid distearoylphosphatidylcholine, cholesterol and the PEG lipid ALC-0159 are mixed and dissolved in ethanol, and the CXCL3 mRNA is encapsulated by a microfluidic device to obtain lipid nanoparticles of CXCL3 mRNA.

[0026] In a second aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the composition of the first aspect and one or more pharmaceutically acceptable carriers.

[0027] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a small molecule drug, a nucleic acid drug antibody, an antibody fragment, a peptide and / or an aptamer for treating liver disease.

[0028] In a third aspect, the present disclosure provides use of the aforementioned mRNA composition or the aforementioned pharmaceutical composition in the preparation of a drug or a drug kit for chemotactic macrophages and / or treating liver diseases.

[0029] In a fourth aspect, the present disclosure provides use of mRNA encoding CXCL3 in the preparation of a drug or a drug kit for chemotactic macrophages and / or treating liver diseases.

[0030] In some embodiments, the medicament or kit comprises a therapeutically effective amount of an mRNA comprising a coding sequence for CXCL3 and a lipid nanoparticle, wherein the mRNA is contained in the lipid nanoparticle.

[0031] In some embodiments, the lipid nanoparticles comprise an ionizable lipid, a non-cationic lipid, cholesterol, and a PEG lipid.

[0032] In some embodiments, the ionizable lipid is selected from (4-hydroxybutyl) aminobis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315, CAS No.: 2036272-55-4), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)aminobis(dodecan-2-ol) (C12-200), 4-(N,N-dimethylamino)butyric acid-6,9,28,31-tetraene-19-triacontanoate (MC3), 1,2-linoleyloxy-N,N-dimethyl-3-aminopropane (DLinD MA), 2,2-linoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2DMA), (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(1H-imidazol-4-yl)propanoic acid ethyl ester (ICE), (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadecadien-9,12-dien-1-yl)tetracosyl-15,18-dien-1-amine (HGT500 0), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecadien-1-yl)tetracosine-4,15,18-triethylenetetramine-1-amine (HGT5001), 3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1-yl]amino]butyl]-2,5-piperazinedione (OF-02), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl ammonium trifluoroacetate (DOSPA), 5-carboxysperminylglycine-bisoctadecylamide (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-(1,2-dimyristoyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybut-4-oxy)-1-(cis, cis-9,1-(cis-1-2-octadeceneoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentyloxy]-3-dimethyl-1-(cis,cis-9',1-2'-octadeceneoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1, 2-N,N'-dilinoleylcarbamyl-3-dimethylpropylamine (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2-((2,3-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethylamine (HGT4003) One or more. In some preferred embodiments, the ionizable lipid is ALC-0315. ,

[0033] In some embodiments, the non-cationic lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimide) In some preferred embodiments, the non-cationic lipid is distearoylphosphatidylcholine.

[0034] In some embodiments, the PEG lipid is selected from one or more of PEG-phospholipids and PEG-ceramides, for example, selected from methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159), dimyristoylglycerol-PEG2000 (DMG-PEG2000), distearoyl phosphatidylethanolamine-PEG2000 (DSPE-PEG2000), dipalmitoyl phosphatidylethanolamine-PEG2000 (DPPE-PEG2000), distearoyl phosphatidylethanolamine-PEG2000 (DMPE-PEG2000), dioleoyl phosphatidylethanolamine-PEG2000 (DOPE-PEG2000), distearoyl phosphatidylethanolamine-PEG1000 (DSPE-PEG1000). 00), dipalmitoylphosphatidylethanolamine-PEG1000 (DPPE-PEG1000), dimyristoylphosphatidylethanolamine-PEG1000 (DMPE-PEG1000), dioleoylphosphatidylethanolamine-PEG1000 (DOPE-PEG1000), distearoylphosphatidylethanolamine-PEG550 (DSPE-PEG550), dipalmitoylphosphatidylethanolamine-PEG550 (DPPE-PEG550), dimyristoylphosphatidylethanolamine-PEG550 (DMPE-PEG550) PEG-cholesterol, PEG2000-ceramide, PEG1000-ceramide, PEG750-ceramide, PEG550-ceramide, one or more thereof. In some embodiments, the PEG lipid is ALC-0159.

[0035] In some embodiments, the lipid nanoparticles comprise ionizable lipid ALC-0315, distearoylphosphatidylcholine, cholesterol, and PEG lipid ALC-0159.

[0036] In some embodiments, the particle size of the lipid nanoparticles is 85-120 nm, for example, it can be 85 nm, 86 nm, 87 nm, 88 nm, 90 nm, 92 nm, 94 nm, 96 nm, 98 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, preferably 88-89 nm, and more preferably 88 nm.

[0037] In some embodiments, the monodispersity of the lipid nanoparticles is 0.030-0.099, for example, it can be 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.063, 0.065, 0.067, 0.070, 0.073, 0.075, 0.077, 0.080, 0.083, 0.085, 0.087, 0.090, 0.095, 0.099, preferably 0.065-0.085, and more preferably 0.075.

[0038] In some embodiments, the zeta potential of the lipid nanoparticles is 3.0-8.0 mV, for example, 3.0 mV, 3.5 mV, 4.0 mV, 4.5 mV, 5.0 mV, 5.3 mV, 5.5 mV, 5.7 mV, 5.9 mV, 6.0 mV, 6.1 mV, 6.3 mV, 6.5 mV, 7.0 mV, 7.5 mV, 8.0 mV, preferably 5.5-6.5 mV, preferably 5.5-6.5 mV, and more preferably 6.1 mV.

[0039] In some embodiments, the amino acid sequence of CXCL3 is as shown in SEQ ID NO:2.

[0040] In some embodiments, the mRNA comprises a CXCL3 coding region, which comprises the nucleotide sequence shown in SEQ ID NO: 5 or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO: 5; in some preferred embodiments, the nucleotide sequence of the CXCL3 coding region is as shown in SEQ ID NO: 5.

[0041] In some embodiments, the mRNA further comprises a 5' cap structure, a 5' UTR, a Kozak sequence, a 3' UTR and / or a poly A region.

[0042] In some embodiments, the 5'UTR sequence is the sequence shown in SEQ ID NO:6.

[0043] In some embodiments, the 3'UTR sequence is the sequence shown in SEQ ID NO:8.

[0044] In some embodiments, the mRNA comprises the nucleotide sequence shown in SEQ ID NO:4 or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO:4; in some preferred embodiments, the nucleotide sequence of the mRNA is as shown in SEQ ID NO:4.

[0045] In some embodiments, the medicament or kit further comprises one or more pharmaceutically acceptable carriers.

[0046] In some embodiments, the medicament or kit further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a small molecule drug, a nucleic acid drug, an antibody, an antibody fragment, a peptide and / or an aptamer for treating liver disease.

[0047] In some embodiments, the dosage of the mRNA is 0.1-1.5 mg / kg each time, for example, it can be 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.5 mg / kg, preferably 0.3-1.2 mg / kg each time, and more preferably 1.0 mg / kg each time.

[0048] In some embodiments, the mRNA composition is administered 1-5 times in one treatment cycle, for example, 1 time, 2 times, 3 times, 4 times, 5 times, preferably 3-4 times, and more preferably 3 times.

[0049] In some embodiments, the liver disease comprises a disease or symptom associated with liver fibrosis; and the liver fibrosis comprises cirrhosis resulting from progressive liver fibrosis.

[0050] Liver (or liver) fibrosis is a complex, progressive process of diffuse excessive deposition of scar tissue in the liver, which is caused by ongoing inflammation and liver cell death that occurs in most types of chronic liver diseases, can cause liver structural damage and changes, and can further evolve into cirrhosis and even liver failure. Many patients with liver diseases, including chronic hepatitis, alcoholic hepatitis, non-alcoholic fatty liver disease, etc., slowly progress to liver fibrosis and cirrhosis after effective treatment through fibrosis repair of the liver. Non-alcoholic fatty liver disease is an umbrella term that includes a continuum of liver conditions that differ in terms of the severity of damage and the resulting fibrosis. Among them, individual hepatic steatosis (fatty liver) is generally referred to as non-alcoholic steatohepatitis, which is usually defined as a more serious process with inflammation and liver cell damage (steatohepatitis), which is generally accompanied by fibrosis and often progresses to cirrhosis. Alcoholic liver disease refers to progressive liver disease caused by excessive, long-term drinking, including alcoholic fatty liver (alcoholic steatosis), alcoholic hepatitis, liver fibrosis and cirrhosis.

[0051] In some embodiments, the liver fibrosis-related diseases or symptoms include chronic hepatitis B, chronic hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease, and diseases or symptoms caused by liver fibrosis. The chronic hepatitis B, chronic hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease progresses or is accompanied by liver fibrosis or cirrhosis. The diseases or symptoms caused by liver fibrosis include hepatorenal syndrome caused by liver fibrosis, liver function metabolic disorders caused by liver fibrosis, primary peritonitis caused by liver fibrosis, autoimmune hepatitis caused by liver fibrosis, primary sclerosing cholangitis caused by liver fibrosis, ascites caused by liver fibrosis, upper gastrointestinal varicose bleeding caused by liver fibrosis and hepatic encephalopathy caused by liver fibrosis; liver fibrosis causes: fatigue, weakness; loss of appetite, nausea, vomiting; chronic indigestion symptoms, such as abdominal distension, constipation or diarrhea, liver pain; chronic gastritis, such as acid reflux, belching, hiccups, upper abdominal pain and upper abdominal fullness, etc. Symptoms: bleeding, chronic hepatitis, such as spider nevi, epistaxis, gingival bleeding, purpura or hemorrhagic spots on the skin and mucous membranes, etc.; infectious complications of liver fibrosis, such as chronic viral hepatitis B, C and D, schistosomiasis, etc.; inborn errors of metabolism, such as Wilson's disease, hemochromatosis, α1-antitrypsin deficiency, etc.; chemical toxicity (such as chronic alcoholic liver disease, chronic drug-induced liver disease) and autoimmune hepatitis, primary biliary cirrhosis and primary, sclerosing cholangitis; liver fibrosis-related: activation of hepatic stellate cells, accumulation of extracellular matrix mediated by activation of hepatic stellate cells, increased serum transaminase levels, inflammatory response of liver tissue, etc.

[0052] In a fifth aspect, the present disclosure provides mRNA encoding CXCL3 and a composition thereof for chemotactic macrophages and / or treating liver fibrosis and / or cirrhosis. The mRNA and the composition thereof are as described above.

[0053] In a sixth aspect, the present disclosure provides a method for chemotactic macrophages and / or treating liver fibrosis and / or cirrhosis, comprising administering to a subject an effective amount of mRNA encoding CXCL3. In some embodiments, the mRNA is located inside a lipid nanoparticle, and the mRNA and the lipid nanoparticle are as described above.

[0054] In some embodiments, the administration comprises delivering an effective amount of the composition, pharmaceutical composition or drug of the present disclosure after introduction into the subject by any route for performing its intended function, and can be administered by any suitable route, including but not limited to intravenous injection, subcutaneous injection or intramuscular injection. In some embodiments, the mRNA, composition, pharmaceutical composition or drug comprising the coding sequence of CXCL3 disclosed herein is administered to the subject in a single dose or multiple doses. In some embodiments, the mRNA, composition, pharmaceutical composition or drug comprising the coding sequence of CXCL3 disclosed herein is administered once, twice, three times, four times, five times, six times or seven times per week, for example, for one month, two months, three months or four months; or once every two weeks, three weeks or four weeks (for example, for six months, ten months or twelve months); or treatment is stopped after continuous administration for one cycle; re-administer a cycle after several months; it can also be used as a chronic treatment, i.e., administered indefinitely.

[0055] One or more technical solutions in the embodiments of the present disclosure have at least the following technical effects or advantages:

[0056] After treating cirrhotic mice with the mRNA-lipid nanoparticle complex prepared by the present disclosure, the level of liver fibrosis in mice was significantly reduced, pseudolobules were improved, liver damage was reduced, and serum ALT and AST decreased. This indicates that the mRNA-lipid nanoparticle complex has potential application value in the delivery of nucleic acid therapeutic preparations in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The denaturing agarose gel electrophoresis results of the prepared CXCL3 mRNA samples are shown.

[0058] Figure 2 Shown is the expression and distribution of luciferase in healthy mice (WT) and mice with liver fibrosis (TAA) injected with luciferase mRNA lipid nanoparticles.

[0059] Figure 3The results of immunofluorescence co-staining of GFP expression and major cell markers in the liver of healthy mice (WT) and liver fibrosis mice (TAA) injected with GFP mRNA lipid nanoparticles are shown.

[0060] Figure 4 Shown is the accumulation of F4 / 80-positive macrophages in the liver of fibrotic mice on the second day after the last injection of lipid nanoparticles (LNP) or CXCL3 mRNA lipid nanoparticle complexes (LNP-CXCL3).

[0061] Figure 5 The results of the mouse liver test 14 days after the last injection of lipid nanoparticles (LNP) or CXCL3 mRNA lipid nanoparticle complexes (LNP-CXCL3) are shown, wherein, Figure 5 A is a general picture of liver tissues in two groups; Figure 5 B is the result of Sirius red staining of liver; Figure 5 C is the result of hydroxyproline analysis in liver tissue.

[0062] Figure 6 The results of the test of mouse serum and major organs (liver, kidney, spleen, lung) 14 days after treatment with CXCL3 mRNA lipid nanoparticle complex are shown, among which, Figure 6 A is a general diagram of liver tissue; Figure 6 B is a general picture of the spleen;

[0063] Figure 6 C is the serum biochemistry (ALT, AST, CREA, UREA) levels of mice; Figure 6 D shows the HE staining results of major organs. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0065] The present disclosure may be implemented in other specific forms without departing from the basic attributes of the present disclosure. It should be understood that, under the premise of no conflict, any and all embodiments of the present disclosure may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present disclosure includes other embodiments obtained by such combination.

[0066] All publications and patents mentioned in this disclosure are hereby incorporated by reference into this disclosure in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0067] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0068] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0069] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0070] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0071] Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0072] As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the disclosure, especially in the claims, may be replaced by the term "consisting of".

[0073] As used herein, the terms "optional", "either", "any", or "any" mean that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. As used in this disclosure, "a" and "an" are used in this disclosure to refer to one or more than one of the grammatical objects.

[0074] As used herein, “and / or” should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0075] As used herein, the term "nucleic acid" includes RNA, DNA and cDNA molecules. It should be understood that due to the degeneracy of the genetic code, a large number of nucleotide sequences encoding a given protein can be generated. "Nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" are used interchangeably and refer to a continuous nucleic acid sequence. The sequence can be a single-stranded or double-stranded DNA or RNA, such as mRNA. The term "messenger RNA (mRNA)" refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA encompasses both modified RNA and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using a recombinant expression system and optionally purified, transcribed in vitro, chemically synthesized, etc. When appropriate, for example, in the case of chemically synthesized molecules, mRNA may include nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. Unless otherwise indicated, mRNA sequences are presented in a 5' to 3' direction.

[0076] The "5' cap structure" mentioned in the present disclosure refers to a special modified structure attached to the 5' end of the mRNA molecule. It is usually composed of a 7-methylguanylate and connected to the 5' end of the mRNA through a triphosphate chain.

[0077] The "poly(A) tail" referred to in the present disclosure is typically a long sequence of cytosine nucleotides, typically about 25 to about 400 adenosine nucleotides, preferably about 50 to about 200 adenosine nucleotides, more preferably about 100 to about 150 adenosine nucleotides, or even more preferably about 120 to about 130 adenosine nucleotides. In a specific embodiment, the poly(A) tail described in the present disclosure contains 125 adenosine nucleotides.

[0078] The 3' untranslated region (3'UTR) referred to in the present disclosure is typically a portion of an mRNA that is located between the protein coding region (i.e., open reading frame) and the poly(A) sequence of the mRNA. The 3'UTR of an mRNA is not translated into an amino acid sequence. The 3'UTR sequence is usually encoded by a gene that is transcribed into the corresponding mRNA during gene expression.

[0079] The 5' untranslated region (5'UTR) mentioned in the present disclosure is typically understood as a specific segment of a messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. Typically, the 5'UTR begins at the transcription start site and ends at a nucleotide before the start codon of the open reading frame. The 5'UTR may include elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements may be, for example, a ribosome binding site or a 5' end oligopyrimidine tract. The 5'UTR may be post-transcriptionally modified, for example, by adding a 5' cap.

[0080] In some embodiments, the mRNA also contains a sequence with one or more regulatory functions. For example, in some specific embodiments, the mRNA also contains a functional sequence that can promote the initiation of translation, such as a Kozak sequence, a Shine-Dalgarno sequence, a TISU (Translation Initiation Stimulation Element) sequence, or an optimized 5'UTR sequence (such as UTR7), etc. For another example, in some specific embodiments, the mRNA also contains a sequence that can regulate the stability of mRNA and the efficiency of translation, such as a Chi-β-globin sequence, an AU enrichment element (AREs) sequence, an internal ribosome entry site (IRESs) sequence, a miRNA binding site (MicroRNA (miRNA) Binding Sites), etc. In the specific implementation, those skilled in the art can confirm the specific sequence and setting position of the above sequence in combination with common sense.

[0081] As used herein, the terms "peptide", "polypeptide", "protein" and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The starting point of a protein or polypeptide is called the "N-terminus" (or amino-terminus, NH2-terminus, N-terminus or amine-terminus), which refers to the free amine (-NH2) group of the first amino acid residue of the protein or polypeptide. The end of a protein or polypeptide is called the "C-terminus" (or carboxyl-terminus, carboxyl-terminus, C-terminus or COOH-terminus), which refers to the free carboxyl group (-COOH) of the last amino acid residue of the protein or peptide.

[0082] As used herein, "nucleic acid construct" refers to an artificially constructed nucleic acid fragment, particularly a DNA fragment, which is intended to be "transplanted" into a target cell (e.g., a bacterial cell) to modify the expression of genomic genes or to express genes / coding DNA sequences that may be included in the construct. The DNA construct may be linear or circular, and a linear or circular DNA construct integrated into the host bacterial genome or expression plasmid is referred to interchangeably herein as an "expression cassette."

[0083] As used herein, the terms "coding region" and "region encoding" and grammatical variations thereof refer to an open reading frame (ORF) of a polynucleotide that produces a polypeptide or protein upon expression.

[0084] As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into polypeptides, the assembly of multiple polypeptides into complete proteins (e.g., enzymes) and / or post-translational modifications of polypeptides or fully assembled proteins (e.g., enzymes). In this article, the terms "expression" and "production" and grammatical equivalents are used interchangeably.

[0085] Although the present disclosure provides some specific amino acid sequences or nucleotide sequences, such as the amino acid sequences or nucleotide sequences shown in the sequence listing, it should be understood that a specific amino acid sequence or nucleotide sequence described in the present disclosure includes conservatively modified variants thereof, such as sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology thereto, as long as the biological function or activity of the specific amino acid sequence or nucleotide sequence is not lost.

[0086] As used herein, the term "homology" has a meaning recognized in the art and is a central concept in comparative biology. The basic meaning of homology is that the two samples being compared (such as an amino acid sequence or a nucleotide sequence) have a common ancestor. In general, if two traits (states) in two species meet any of the following two conditions, the two traits can be called a pair of homologous traits: 1. They are the same as a trait found in the ancestral group of these species; 2. They are different traits with an ancestor-descendant relationship. The homology (%) of nucleotide or amino acid sequences can be determined using programs commonly used in the field (such as BLAST) according to the initial settings. As used herein, the term "homology" refers to the exact nucleotide-nucleotide or amino acid-amino acid correspondence of two polynucleotides or amino acid sequences, respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "homology (%)". Whether it is a nucleic acid or an amino acid sequence, the homology (%) of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100.

[0087] The term "conservatively modified variants" is used to refer to amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids encoding the same or conservatively modified amino acid sequence variants. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, it is well known in the art that degenerate codons (and therefore corresponding DNA codons, T replacing U) can be used interchangeably to encode each specific amino acid, and a degenerate codon refers to a different three-letter codon used to specify the same amino acid, for example, the codons GCA, GCC, GCG and GCU all encode alanine. Therefore, at any position where alanine is specified by a codon, the codon can be changed to any corresponding codon without changing the encoded polypeptide. This nucleic acid variation is a "silent variation", which is a conservatively modified variation. The skilled person will recognize that each codon in a nucleic acid (except AUG and UGG, AUG is usually only a codon for methionine, and UGG is only a codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of a nucleic acid encoding a polypeptide of the present disclosure is unambiguous in each of the described polypeptide sequences, which is included in the scope of the present disclosure.

[0088] For amino acid sequences, the skilled artisan will recognize that each substitution, deletion or addition of a nucleic acid, peptide, polypeptide or protein sequence that changes, adds or deletes a single amino acid or a small number of amino acids in the coding sequence is a "conservatively modified variant", wherein the change results in the replacement of an amino acid with a chemically similar amino acid. Thus, an amino acid residue selected from any integer between 1-15 may be altered. For example, 1, 2, 3, 4, 5, 7 or 10 changes may be produced. Conservatively modified variants typically provide biological activity similar to that of the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzymatic activity or ligand / receptor binding is typically at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of that of a native protein with its native substrate. It is well known to those skilled in the art to provide a table of conservative substitutions of amino acids that are functionally similar. For example, modifications may be introduced into the sequence listing described herein by standard techniques known in the art (e.g., gene synthesis and PCR-mediated mutagenesis). Conservative modifications include conservative amino acid substitutions, where a "conservative substitution" is one in which an amino acid is substituted by another amino acid with similar properties, such as replacement with an amino acid with a similar side chain, whereby a person skilled in the art of peptide chemistry would expect that the secondary structure and hydrophilic properties of the polypeptide are substantially unchanged. Families of amino acid residues with similar side chains are already defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0089] As used herein, the term " lipid nanoparticles " (lipid nanoparticles), " LNP " and " LNPs " are used interchangeably, and the lipid nanoparticles can comprise any lipid that can form the particle that one or more nucleic acid molecules are attached to or one or more nucleic acid molecules are encapsulated therein.In this article, the lipid nanoparticles that one or more nucleic acid molecules are encapsulated therein can be referred to as nucleic acid lipid nanoparticle compositions, also can be referred to as nucleic acid lipid nanoparticle complexes.Term " lipid " refers to a group of organic compounds, and they are derivatives (such as esters) of fatty acids and feature being generally insoluble in water but soluble in many organic solvents.The lipid nanoparticles refers to any particle or vesicle with nanometer size formed by one or more lipid components.

[0090] As used herein, the terms "ionizable lipid", "cationic lipid" and "ionizable cationic lipid" are used interchangeably and refer to lipid molecules that are capable of being positively charged under physiological pH conditions. Cationic liposomes are easily bound to negatively charged nucleic acids, i.e., interact with negatively charged phosphate groups present in nucleic acids through electrostatic forces.

[0091] As used herein, the terms "non-cationic lipids" and "neutral lipids" are used interchangeably and refer to lipids that are uncharged or exist in a zwitterionic form at a selected pH value. Non-cationic lipids may regulate nanoparticle fluidity by promoting lipid phase transition to a lipid bilayer structure and improve efficiency, and may also affect the specificity of the target organ.

[0092] As used herein, the term "polyethylene glycol lipid" (PEG lipid) refers to a lipid molecule comprising a lipid portion and a polyethylene glycol portion.

[0093] Various methods of lipid nanoparticle preparation are suitable for the present disclosure. For example, ethanol dilution, freeze-thaw, thin film hydration, sonication, extrusion, high pressure homogenization, detergent dialysis, microfluidization, tangential flow diafiltration, sterile filtration and / or freeze drying can be used.

[0094] As used herein, the term "particle size" or "particle diameter" refers to the average diameter of particles in a sample measured by dynamic light scattering (DLS), multi-angle light scattering (MALS), nanoparticle tracking analysis or similar techniques. It should be understood that the dispersion of lipid nanoparticles described herein is not uniformly sized, but can be described by the average diameter and an optional polydispersity index.

[0095] As used herein, the term "encapsulation efficiency" refers to the ratio of nucleic acids encapsulated inside lipid nanoparticles to all nucleic acids. Encapsulation efficiency is usually detected by fluorescence spectrophotometry. Taking RNA as an example, the amount of RNA free outside the lipid nanoparticles in the LNP-RNA solution is first detected with RiboGreen fluorescent dye, and then Triton-100 is used to destroy the structure of the lipid nanoparticles, so that the RNA is released into the external solution, and the total amount of RNA in the solution is detected. The difference between the two is the amount of RNA encapsulated inside the LNP particles, thereby obtaining the encapsulation efficiency.

[0096] As described herein, the term "Zeta potential" refers to the total surface charge obtained by a nanoparticle in a specific medium (e.g., water), which is a measure of electrostatic attraction and repulsion. Zeta potential values ​​indicate dispersion stability, aggregation, and diffusion behavior. Zeta potential can be calculated based on electrokinetic data obtained from, for example, laser Doppler velocimetry. In this technique, a voltage is applied to a pair of electrodes at both ends of a pool containing a nanoparticle dispersion. Charged nanoparticles are attracted to electrodes with opposite charges, and their speed is measured and expressed as their electrophoretic mobility per unit field strength. Zeta values ​​can predict the penetration of various cell membranes. The zeta potential of lipid nanoparticles can be measured by Zetasizer Pro (an instrument from Malvern Instruments Ltd). LNP can be diluted in a pH buffer (e.g., PBS pH=7.4) to a certain level (e.g., 1.0 ng / μL) of total mRNA and then loaded into a disposable folded capillary pool (e.g., DTS1070). The sample can be balanced for a period of time (e.g., 120 seconds), and the measurement is repeated multiple times, with a period of time (e.g., 20 seconds) between measurements.

[0097] As used herein, the term "monodispersity" refers to the uniformity of the distribution of particles or grains in size, shape and structure. If a nanoparticle system has a very narrow particle size distribution, that is, most of the particles are almost equal in size, then the system is considered to be monodisperse. A high monodispersity index means that the particle size is more consistent.

[0098] As used herein, the term "encapsulate" means to enclose, surround, or encompass.

[0099] As used herein, the term "delivery" refers to providing an entity to a target. For example, delivering a polynucleotide to a subject may involve administering a nanoparticle composition comprising the polynucleotide to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

[0100] As used herein, the term "chemotaxis" refers to the positive (toward the stimulus) or negative (away from the stimulus) migration of cells induced by chemical stimulation, especially refers to the positive migration. The chemotaxis of macrophages refers to the migration of macrophages induced by chemical stimulation.

[0101] As used herein, the term "aptamer" refers to a polynucleotide, typically RNA or DNA, that has useful biological activity in terms of biochemical activity, molecular recognition or binding properties. Typically, an aptamer has a molecular activity such as binding to a target molecule at a specific epitope (region).

[0102] As used herein, the term "treatment" is intended to include prevention and treatment, methods by which beneficial or desired results, including clinical results, can be obtained through treatment. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dose of one or more other drugs required to treat the disease, delaying the progression of the disease, improving or improving the quality of life, increasing weight gain and / or prolonging survival. "Treatment" also includes the reduction of pathological results (e.g., tumor volume) of cancer. In the context of cancer, "treatment" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing overall tumor burden, and improving one or more symptoms associated with the disease.

[0103] As used herein, the term "effective amount" or "therapeutically effective amount" encompasses an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0104] As used herein, the terms "subject", "individual" or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, apes, humans, farm animals, competitive animals, and pets.

[0105] The amount of the drug administered may depend on the subject being treated, the subject's age, health, sex, and weight, the type of concurrent treatment (if any), the severity of the condition, the nature of the desired effect, the manner and frequency of treatment, and the judgment of the prescribing physician. The frequency of administration may also depend on the pharmacodynamic effect on arterial oxygen tension. However, the most preferred dosage may be adjusted for individual subjects, as will be understood by those skilled in the art and can be determined without undue experimentation. This generally includes adjusting the standard dosage (e.g., if the patient is of low weight, then reducing the dosage).

[0106] As used herein, the term "pharmaceutical composition" refers to a mixture of one or more components. The purpose of a pharmaceutical composition is to facilitate administration of a compound of the present disclosure to an organism. The pharmaceutical composition of the present disclosure may contain one or more pharmaceutically acceptable salts, pharmaceutically acceptable carriers and / or adjuvants, such as antioxidants, preservatives, wetting agents, emulsifiers and dispersants, etc.

[0107] As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation adjuvant or carrier conventional in the art for use with a therapeutic agent, which together constitute a "pharmaceutical composition" for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation employed. Suitable carriers are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, liposomes, polymer micelles or inorganic nanocarriers, etc.

[0108] The "pharmaceutical composition" of the present disclosure can also be administered to patients or subjects in need of such treatment in any suitable manner, such as oral, intravenous, parenteral, rectal, pulmonary or topical administration. When used for oral administration, the pharmaceutical composition can be prepared into oral preparations, such as oral solid preparations, such as tablets, capsules, pills, granules, etc.; or, oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When prepared into oral preparations, the pharmaceutical preparations may also contain suitable fillers, binders, disintegrants, lubricants, etc.

[0109] The pharmaceutical composition of the present invention can be prepared into any pharmaceutically acceptable dosage form for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration, for example, it can be formulated into tablets, lozenges, capsules, pills, solutions, suspensions, syrups, injections, suppositories, inhalants or sprays.

[0110] As used herein, the term "kit" refers to one or more components of a package, which can be packaged separately or placed in a container, such as a tube, bottle, vial, bag, blister, syringe or other suitable container device. The kit described herein may also include instructions for implementing the subject method using the components of the kit (e.g., instructions for preparing a pharmaceutical composition described herein and / or instructions for using a pharmaceutical composition). The instructions for implementing the subject method are usually recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Therefore, the instructions can be present in the kit in the form of a package insert, in the label of the container of the kit or its components (i.e., components associated with the package or small package). In some embodiments, the instructions exist as an electronic storage data file present in a suitable computer-readable storage medium (e.g., CD-ROM, disk, etc.). In other embodiments, the actual instructions are not present in the kit, but are provided for obtaining instructions from a remote source, such as through the Internet. An example of this embodiment is a kit including a website where the instructions can be viewed and / or from which the instructions can be downloaded.

[0111] As used herein, the terms "fibrosis" and "fibrotic condition" are used interchangeably herein and refer to pathological tissue healing in which connective tissue replaces normal parenchymal tissue, resulting in considerable tissue remodeling and the formation of permanent scar tissue. Fibrosis may occur in any of a number of organs, including but not limited to the liver, lungs, kidneys, skin, eyes, or myocardium. As used herein, the terms fibrosis or fibrotic condition also refer to different stages of tissue fibrosis.

[0112] As used herein, the term "liver disease (hepatopathy)" refers to pathological changes that occur in the liver. The term "hepatitis cirrhosis" is also known as cirrhosis, which refers to the formation of fibrous tissue (fibrosis) in the liver, replacing dead liver cells in the liver. Cirrhosis is an end-stage liver disease characterized by damage caused by liver disease, which leads to the formation of fibrotic scar tissue and causes impaired liver function. Liver damage causes tissue repair and subsequent scar tissue formation. Over time, scar tissue replaces normal functioning tissue, leading to decreased liver function and eventually developing into cirrhosis.

[0113] As used herein, the term "therapeutic agent" refers to an agent that has a therapeutic, diagnostic and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject.

[0114] As used herein, the term "CXCL3" refers to any native CXCL3 (chemokine (CXC motif) ligand 3; also known as macrophage inflammatory protein 2-β (MIP2-β)) from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CXCL3 as well as any form of CXCL3 derived from processing in a cell. The term also encompasses naturally occurring variants of CXCL3, such as splice variants or allelic variants.

[0115] In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure is further described in detail below in conjunction with the examples. If the specific conditions are not specified in the examples, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present disclosure, numerous specific details are given in the specific embodiments below. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure.

[0116] Example

[0117] Example 1. Preparation of CXCL3 mRNA lipid nanoparticles (CXCL3 mRNA-LNP)

[0118] 1. CXCL3 mRNA synthesis:

[0119] ① Obtain the DNA sequence of mouse (mus musculus) CXCL3 from NCBI (https: / / www.ncbi.nlm.nih.gov / ).

[0120] CXCL3 DNA sequence (SEQ ID NO: 1) as follows: ATGGCCCCTCCCACCTGCCGGCTCCTCAGTGCTGCACTGGTCCTGCTGCTGCTGCTGGCCACCAACCACCAGGCTACAGGGGCTGTTGTGGCCAGTGAGCTGCGCTGTCAGTGCCTGAACACCCTACCAAGGGTTGATTTTGAGACCA TCCAGAGCTTGACGGTGACGCCCCCAGGACCCACTGCACCCAGACAGAAGTCATAGCCACTCTCAAGGATTGGTCAAGAAGTTTGCCTCAACCCCCAAGGCCCCAGGCTTCAGATAATCATCAAGAAGATACTGAAGAGCGGCAAGTCCAGCTGA.

[0121] The CXCL3 amino acid sequence (SEQ ID NO: 2) is as follows: MAPPTCRLLSAALVLLLLLATNHQATGAVVASELRCQCLNTLPRVDFETIQSLTVTPPGPH CTQTEVIATLKDGQEVCLNPQGPRLQIIIKKILKSGKSS.

[0122] ② Design the expression cassette of CXCL3 mRNA. From the 5' end to the 3' end, it is: 5'UTR, "Kozak sequence + Cxcl3 DNA sequence", 3'UTR, poly A and BspQI restriction enzyme cleavage site. The nucleotide sequence is as follows:

[0123] CCAGATCTCACCACAGCCCTTCGCACCAGCTCCCGCGCTCCAGACTCCAGCCACACT

[0124] CCAGCCTAGCCGCCAGGCACCATGGCCCCTCCCACCTGCCGGCTCCTCAGTGCTGCAC

[0125] TGGTCCTGCTGCTGCTGCTGGCCACCAACCACCAGGCTACAGGGGCTGTTGTGGCCA

[0126] GTGAGCTGCGCTGTCAGTGCCTGAACACCCTACCAAGGGTTGATTTTGAGACCATCC

[0127] AGAGCTTGACGGTGACGCCCCCAGGACCCCACTGCACCCAGACAGAAGTCATAGCC

[0128] ACTCTCAAGGATGGTCAAGAAGTTTGCCTCAACCCCCAAGGCCCCAGGCTTCAGATA

[0129] ATCATCAAGAAGATACTGAAGAGCGGCAAGTCCAGCTGAGCCGGGGAAAGGAGGAA

[0130] GCCCCTCACCATGATGAAGGACCTGCATTCTAAATCAGAGAAAGCGATCCATCCCA

[0131] ACGGTGTCTGGATGTGTCTTTACCTGCTTGTTTTAACAACTCCTGAGAGTTCATACCT

[0132] ATTTTATTTCACATGTAAAATAACTACGGGTATAATTGCATCTACTTGCAAGATGGCATC

[0133] TTATTTAATGTTTATTTAATATCAAGTCTAAGTTTAACCAAGCCTATTCTTAACTTGAAG

[0134] GTTAGAGATTTCAGATTCTGTTAGGGAGTTATATTGCTATTTCTGAGGGCAGTATTCCTT

[0135] GGCTGGCCATTTTGGTGGCAGCTGTGATAGTCAAAAAACAGACAAACAAGGTCATGC

[0136] CGGTGTAGGAAGAATGCATGTGCACATCTAGTTTTGTAACTATACAAGATTATCAGTTG

[0137] TTATTTATTGAAATGGTCTTACGGTGTCACATGAAATATCAACTTTGTGTTAAAAGCTTT

[0138] AAGAATTATAATGCATTATATATTTCTTAGACATTTTTAAGTGATTTTTTAAAAATAAGGC

[0139] ACAGTGACTATTTAATGCCTTTATGTCTTAGAATGGAGAAGTTTAAATATTTATTGGTAT

[0140] TTTTACAAATAATTTGAAAATAAAATATTTTAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCTCTTC (SEQ ID NO: 3).

[0141] ③ Commissioned Sangon Biotechnology to synthesize the above sequence and clone it into the pUC57 vector. After sequencing confirmation, the in vitro transcription template was successfully obtained and used T7 RNA synthesis kit (NEB, E2050S) was used for in vitro transcription. ARCA (NEB, S1411S) was added to cap the cells during the process. Finally, 5 M LiCl solution was added to mix the cells and placed at -20°C for 30 minutes. The precipitate was collected. The precipitate was washed with 70% ethanol to remove impurities. Finally, the purified mRNA was dissolved in enzyme-free water to obtain the CXCL3 mRNA sample. The sample was detected by denaturing agarose gel electrophoresis. Figure 1 The size of the obtained mRNA was consistent with the expected one, and CXCL3 mRNA was successfully prepared.

[0142]

[0143] Among them, the mRNA sequence (SEQ ID NO: 5) of the CXCL3 coding region is as follows: AUGGCCCCUCCCACCUGCCGGCUCCUCAGUGCUGCACUGGUCCUGCUGCUGCUGCUGGCCACCAACCACCAGGCUACAGGGGCUGUUGUGGCCAGUGAGCUGCGCUGUCAGUGCCUGAACACCCUACCAAGGGUUGAUUUUGAGACCAUCCAGAGCUUGACGGUGACGCCCCCAGGACCCCACUGCACCCAGACAGAAGUCAUAGCCACUCUCAAGGAUGGUCAAGAAGUUUGCCUCAACCCCCAAGGCCCCAGGCUUCAGAUAAUCAUCAAGAAGAUACUGAAGAGCGGCAAGUCCAGCUGA.

[0144] The 5' cap is the CAP1 structure.

[0145] The 5' UTR sequence (SEQ ID NO: 6) is as follows:

[0146] CCAGAUCUCACCACAGCCCUUCGCACCAGCUCCCGCGCUCCAGACUCCAGCCACACUCCAGCCUAGCGCCA.

[0147] The Kozak sequence (SEQ ID NO: 7) is as follows:

[0148] GGCACC.

[0149] The 3' UTR sequence (SEQ ID NO: 8) is as follows:

[0150] GCCGGGGAAAGGAGGAAGCCCCUCACCAUGAUGAAGGACCUGCAUUCUAAAUCAG

[0151] AGAAAAGCGAUCCAUCCCAACGGUGUCUGGAUGUGUCUUUACCUGCUUGUUUUU

[0152] AACAACUCCUGAGAGUUCAUACCUAUUUUAUUUCACAUGUAAAAUAACUACGGG

[0153] UAUAAUUGCAUCUACUUGCAAGAUGGCAUCUUAUUUAAUGUUUAUUUAAUAUCA

[0154] AGUCUAAGUUUAACCAAGCCUAUUCUUAACUUGAAGGUUAGAGAUUUUCAGAUU

[0155] CUGUUAGGGAGUUAUAUUGCUAUUUCUGAGGCAGUAUUCCUUGGCUGGCCAUUU

[0156] UGGUGGCAGCUGUGAUAGUCAAAAAACAGACAAACAAGGUCAUGCCGGUGUAGG

[0157] AAGAAUGCAUGUGCACAUCUAGUUUUGUAACUAUACAAGAUUAUCAGUUGUUAU

[0158] UUAUUGAAAUGGUCUUACGGUGUCACAUGAAAUAUCAACUUUGUGUUAAAGCUU

[0159] UAAGAAUUAUAAUGCAUUAUAUAUUUCUUAGACAUUUUAAGUGAUUUUUUAAAA

[0160] AUAAGGCACAGUGACUAUUUAAUGCCUUUAUGUCUUAGAAUGGAGAAGUUUAAAUAUUUAUUGGUAUUUUUACAAAUAAUUUGAAAAUAAAAUAUUUUAAAAUAA。

[0161] The poly-A sequence (SEQ ID NO: 9) is as follows: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAA.

[0162] 2. Encapsulation:

[0163] The CXCL3 mRNA obtained above was dissolved in citrate buffer to a final concentration of 0.5 mg / mL, and the ionizable lipid ALC-0315, distearoylphosphatidylcholine, cholesterol, and PEG lipid ALC-0159 were mixed and dissolved in ethanol at a ratio of 50%: 10%: 38.5%: 1.5% to obtain a lipid mixture with a final concentration of 10 mM, and then the mRNA and the lipid mixture were quickly mixed through a nanoparticle preparation system (NanoAssembler Ignite, Cytiva), with a flow rate ratio of 3: 1 and a total flow rate of 8 mL / min. After mixing, the obtained product was ultrafiltered to remove excess ethanol, and finally lipid nanoparticles (CXCL3 mRNA-LNP) encapsulated with CXCL3 mRNA were obtained.

[0164] The particle size, monodispersity and zeta potential of the lipid nanoparticles obtained above were measured using a laser particle size analyzer (Nano ZS, Malvern). At the same time, the lipid nanoparticles were lysed with 0.2% Triton X-100, and the mRNA encapsulation efficiency was measured using the Quant-iTRiboGreen RNA kit (R11490, Thermo Fisher). The characterization results are as follows:

[0165] Particle size: 88.52 ± 0.459 nm;

[0166] Monodispersity: 0.0749 ± 0.010138;

[0167] ζ potential: 6.101±0.3981mV;

[0168] mRNA encapsulation efficiency: 94.33%.

[0169] Example 2. Targeted detection of lipid nanoparticles (LNP)

[0170] 1. Targeted detection in liver fibrosis

[0171] C57 mice were fed with 250mg / L TAA (thioacetamide) in drinking water for a total of 8 weeks to construct a liver fibrosis model. In this experiment, the mRNA contained in LNP is Luciferase mRNA that can express luciferase. The preparation method of Luciferase mRNA-LNP is the same as in Example 1, except that CXCL3 mRNA is replaced with Luciferase mRNA. Healthy mice were used as a control group, and 40μg Luciferase mRNA-LNP was injected into the tail vein of mice with liver fibrosis, and the bioluminescence of healthy mice and liver fibrosis mouse tissues was photographed by small animal in vivo imaging technology 8 hours after injection. 150mg / kg of D-luciferin was injected intraperitoneally 10 minutes before shooting. After the mice were anesthetized with isoflurane, the distribution of mRNA delivered by LNP in the two groups of mice was observed by Tianneng small animal in vivo imaging instrument.

[0172] After Luciferase mRNA-LNP enters the mouse and expresses luciferase, the mouse is given a luciferin substrate. Under the catalysis of luciferase, the substrate emits light, and the small animal in vivo imaging instrument detects bioluminescence to reflect the expression and distribution of luciferase. This detection method can specifically determine the distribution and expression of mRNA.

[0173] The results are as follows Figure 2 As shown, the mRNA delivered by LNP was mainly expressed in the mouse liver. The liver fibrosis background did not affect the location of its delivery and expression, as well as the expression efficiency.

[0174] 2. Cellular location of target protein expression in fibrotic liver

[0175] In this experiment, the mRNA contained in LNP is GFP mRNA that can express green fluorescence (Green fluorescent protein, GFP). The other experimental conditions and steps are the same as 2.1, only Luciferase mRNA is replaced by GFP mRNA. The main cell markers (Hnf4α, CK19, CD31, F4 / 80, α-sma) and GFP expression positions in the mouse liver are detected by immunofluorescence co-staining technology. The specific cells expressing GFP mRNA in the liver of the control group and fibrosis group mice can be specifically determined.

[0176] The results are as follows Figure 3 As shown, LNP-delivered mRNA was mainly expressed in mouse hepatocytes and Kupffer cells, and the liver fibrosis background did not affect the location or efficiency of its expression.

[0177] Example 3. CXCL3 mRNA-LNP for the treatment of liver cirrhosis

[0178] 1. A liver cirrhosis model was constructed by TAA drinking water. The modeling method adopted was: 250 mg / L TAA was added to daily drinking water for a total of 8 weeks. The drug was administered by tail vein injection, with equal volumes of CXCL3 mRNA-LNP or blank LNP. CXCL3 mRNA-LNP and blank LNP were obtained according to the preparation method of Example 1, except that blank LNP did not encapsulate mRNA. The dosage and administration time are shown in Table 1. Healthy mice were used as the control group, with normal drinking water and no administration.

[0179] Table 1. Dosage regimen and test of each group of mice in Example 3

[0180]

[0181] 2. 48 hours after the last administration or 14 days after recovery, serum and organ samples (liver, spleen, lung, and kidney) of each group were collected, and liver tissue was stained with HE and picrosirius red, and immunohistochemistry was used to detect the macrophage marker F4 / 80. A kit (elabscience, E-BC-K062-M) was used to detect liver tissue hydroxyproline to evaluate whether macrophages were accumulated in the liver and whether the fibrosis level was relieved and recovered after administration.

[0182] The results are analyzed as follows:

[0183] like Figure 4 It shows that a large number of F4 / 80-positive macrophages accumulated in the liver of mice with liver fibrosis on the second day after the last injection of CXCL3 mRNA-LNP.

[0184] like Figure 5 The results showed that the granularity of the liver surface of treated mice was reduced, and picrosirius red staining and hydroxyproline analysis showed that the level of intrahepatic fibers was significantly reduced.

[0185] Example 4. Safety of CXCL3 mRNA-LNP

[0186] Healthy mice were divided into two groups, 3 mice in each group, and were injected with 1 mg / kg blank LNP (WT) or 1 mg / kg CXCL3 mRNA-LNP (WT+LNP-CXCL3) by tail vein. The preparation methods of blank LNP and CXCL3 mRNA-LNP were the same as in Example 3. The mice were given 3 times, each time interval was 24 hours, and the serum and organs of the mice were collected 14 days after the last injection. The detection method was the same as in Example 3. In addition, a third-party testing agency (Lilai Bio) was commissioned to test serum biochemistry (ALT, AST, CREA, UREA).

[0187] like Figure 6HE staining results of mouse liver, kidney, spleen and lung showed that CXCL3 mRNA-LNP did not cause organ abnormalities in mice. Serum ALT, AST, CREA and UREA results showed no damage to the liver and kidney functions of mice.

Claims

1. An mRNA composition comprising mRNA and lipid nanoparticles, wherein the mRNA is contained in the lipid nanoparticles, and the mRNA composition is characterized in that the mRNA is capable of expressing CXCL3.

2. The mRNA composition according to claim 1, wherein The amino acid sequence of CXCl3 is shown in SEQ ID NO: 2; Preferably, the mRNA comprises a CXCL3 coding region, the CXCL3 coding region comprises the nucleotide sequence shown in SEQ ID NO: 5 or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO: 5; more preferably, the nucleotide sequence of the CXCL3 coding region is as shown in SEQ ID NO: 5; Preferably, the mRNA further comprises a 5' cap structure, a 5'UTR, a Kozak sequence, a 3'UTR and / or poly A; Preferably, the 5'UTR sequence is the sequence shown in SEQ ID NO: 6; Preferably, the 3'UTR sequence is the sequence shown in SEQ ID NO: 8; Preferably, the mRNA comprises the nucleotide sequence shown in SEQ ID NO:4 or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO:4; more preferably, the nucleotide sequence of the mRNA is as shown in SEQ ID NO:

4.

3. The mRNA composition according to claim 1 or 2, wherein the lipid nanoparticles comprise ionizable lipids, non-cationic lipids, cholesterol and PEG lipids; Preferably, the ionizable lipid is selected from one or more of ALC-0315, C12-200, MC3, DLinDMA, DLinKC2DMA, ICE, HGT5000, HGT5001, OF-02, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DOTMA, DLenDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, DLin-K-XTC2-DMA, and HGT4003, more preferably ALC-0315; Preferably, the non-cationic lipid is selected from one or more of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylethanolamine, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, more preferably distearoylphosphatidylcholine; Preferably, the PEG lipid is selected from one or more of PEG-phospholipids and PEG-ceramide; More preferably, the PEG lipid is selected from one or more of ALC-0159, DMG-PEG2000, DSPE-PEG2000, DPPE-PEG2000, DMPE-PEG2000, DOPE-PEG2000, DSPE-PEG1000, DPPE-PEG1000, DMPE-PEG1000, DOPE-PEG1000, DSPE-PEG550, DPPE-PEG550, DMPE-PEG550, PEG-cholesterol, PEG2000-ceramide, PEG1000-ceramide, PEG750-ceramide, PEG550-ceramide, more preferably ALC-0159; Preferably, the lipid nanoparticles comprise the ionizable lipid ALC-0315, distearoylphosphatidylcholine, cholesterol and the PEG lipid ALC-0159.

4. The mRNA composition according to any one of claims 1 to 3, wherein The particle size of the lipid nanoparticles is 85-120 nm, preferably 88-89 nm, more preferably 88 nm; Preferably, the monodispersity of the lipid nanoparticles is 0.030-0.099, more preferably 0.065-0.085, and further preferably 0.

075.

5. A pharmaceutical composition comprising a therapeutically effective amount of the mRNA composition according to any one of claims 1 to 4 and one or more pharmaceutically acceptable carriers; Preferably, the pharmaceutical composition further comprises an additional therapeutic agent; Preferably, the additional therapeutic agent comprises a small molecule drug, a nucleic acid drug, an antibody, an antibody fragment, a peptide and / or an aptamer for treating liver disease.

6. Use of mRNA encoding CXCL3 in the preparation of a drug or a drug kit for treating liver diseases.

7. The use according to claim 6, wherein: The amino acid sequence of CXCL3 is shown in SEQ ID NO: 2; Preferably, the mRNA comprises a CXCL3 coding region, the CXCL3 coding region comprises the nucleotide sequence shown in SEQ ID NO: 5 or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO: 5; more preferably, the nucleotide sequence of the CXCL3 coding region is as shown in SEQ ID NO: 5; Preferably, the mRNA further comprises a 5' cap structure, a 5'UTR, a Kozak sequence, a 3'UTR and / or poly A; Preferably, the 5'UTR sequence is the sequence shown in SEQ ID NO: 6; Preferably, the 3'UTR sequence is the sequence shown in SEQ ID NO: 8; Preferably, the mRNA comprises the nucleotide sequence shown in SEQ ID NO:4 or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO:4; more preferably, the nucleotide sequence of the mRNA is as shown in SEQ ID NO:

4.

8. Use of the mRNA composition according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a drug or a medicine kit for treating liver diseases.

9. The use according to any one of claims 6 to 8, wherein: The dosage of the mRNA is 0.1-1.5 mg / kg each time, preferably 0.3-1.2 mg / kg each time, and more preferably 1.0 mg / kg each time.

10. The use according to any one of claims 6 to 9, wherein: The liver disease includes diseases or symptoms related to liver fibrosis; the liver fibrosis includes liver cirrhosis caused by liver fibrosis; Preferably, the liver fibrosis-related diseases or symptoms include chronic hepatitis B, chronic hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease and diseases or symptoms caused by liver fibrosis.

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